Agents that are effective in killing neoplastic and other diseased or abnormal cells generally cannot be administered to a patient in effective doses because they also exert cytotoxic effects on normal cells. Therapeutic protocols for treating cancer and other disorders using cytotoxic agents such as toxins, drugs, radioisotopes, and the like are generally limited by the toxicity of the cytotoxic agent to normal cells and tissues. Efforts have therefore been directed to linking cytotoxic therapeutic agents to targeting agents, such as antibodies, which are capable of binding to certain target cells and tissue(s).
Research efforts in the field of tumor immunology have identified antibodies to antigenic determinants expressed preferentially on tumor cells. Such antibodies, or fragments thereof, may be employed as carriers for cytotoxic agents to provide selective delivery of cytotoxic agents to target tumor tissues. Therapeutic immunoconjugates comprising an active moiety exhibiting therapeutic properties and a targeting agent exhibiting specificity and affinity for target cells, tissue(s), antigens, or the like are believed to be of tremendous potential in the treatment of cancer and a variety of other diseases.
One type of conjugate thought to be potentially useful in the treatment of diseases such as cancer is an antibody-toxin conjugate consisting of a bacterial or plant toxin, or some portion thereof, covalently linked to an antibody. Initially, such immunoconjugates comprised reduced and sulfhydryl activated polyclonal antibodies linked to reduced toxins. This method of conjugation was relatively uncontrolled and unpredictable, since antibody and toxin disulfide bonds could reform as readily as antibody-toxin hybrids. Heterobifunctional reagents having two different reactive end groups were subsequently developed and became preferred compounds for linking antibodies to toxins. Using heterobifunctional reagents, the number of free sulfhydryl groups available for conjugation, and ratios of antibody to toxin in the immunoconjugate product, could be controlled fairly effectively. The presence of two different reactive groups on heterobifunctional reagents permitted more directed, predictable, and reproducible linkage of targeting agents to cytotoxic agents. Numerous heterobifunctional reagents have been reported in the literature for derivatizing antibody and/or toxin molecules.
Coupling the targeting agent of an immunoconjugate to a toxin moiety through disulfide bonds which can be reductively cleaved may be achieved employing various heterobifunctional reagents. Using other heterobifunctional reagents, targeting and active moieties may be covalently linked through bonds that are not affected by reducing agents, such as amide or thioether bonds. Disulfide-bonded toxin immunoconjugates were initially believed to be necessary to mimic the disulfide linkage of A and B chains of native toxin. It was thought that the native disulfide linkage had to be reductively cleaved to liberate the active A chain of the toxin molecule within the cell. Linkage of A chains of toxins with antibodies through non-reducible bonds generally produced immunoconjugates of decreased potency. Immunoconjugates of Pseudomonas exotoxin (PE) coupled to monoclonal antibodies (MAbs) or to epidermal growth factor (EGF) are disclosed in U.S. Pat. No. 4,545,985 (I. Pastan et al., 1985).
The serum half-life of therapeutic agents such as immunoconjugates may directly impact and may be related to numerous factors affecting the efficiency of therapeutic protocols. Enhancement of serum half-life is an important objective since, in general, the longer therapeutic agents remain in circulation, the higher the likelihood they will reach target cells and tissue(s).
Antibody fragments may offer advantages as the targeting component in immunoconjugate compounds since antibody fragments accumulate at target sites, such as tumor sites, more rapidly than their whole antibody counterparts. Rapid localization may be due to the smaller size of the fragments. In addition, decreased fragment size may facilitate egress from circulation across the blood vessel and capillary walls into the tumor bed. However, the smaller fragments generally have shorter serum half-lives than whole antibody, and the increased rapidity of target tissue localization may not offset the reduced serum half-life characteristics of the immunoconjugates. That is, conjugates comprising antibody fragments may be cleared prior to tumor localization, despite their increased tumor localization capability. Thus, it would be desirable to prolong serum half-life of conjugates comprising relatively small targeting moieties, such as antibody fragments, to take advantage of their target site localization characteristics.
Moreover, clearance of therapeutic agents such as immunoconjugates from circulation is generally harmful, since high concentrations of the active (typically cytotoxic) moiety accumulate in non-target tissues and destroy tissue at those non-target sites. Still further, when significant quantities of therapeutic agents are removed from circulation before they reach the target site(s), higher doses are required to produce the desired diagnostic or therapeutic effect. Higher doses in turn cause more damage to normal tissues and generally cannot be tolerated due to the accompanying adverse side effects.
Providing enhanced serum stability of therapeutic agents is another important objective. Immunoconjugates comprising an active moiety bound to a targeting moiety must remain stably bound in serum for sufficient time to allow the targeting moiety to selectively deliver the active moiety to the target site. If the bond(s) linking the active and targeting moieties are not sufficiently stable, the targeting moiety may become detached from the active moiety before it has reached the target site, thereby contributing to non-target retention of the active agent and resultant toxicity.
Effective doses of therapeutic drugs, such as anti-cancer drugs, typically require administration of fairly large quantities of drug. In many cases, it is difficult to administer an effective dose of a therapeutic drug unless multiple molecules of the drug are bound to each carrier moiety. Binding multiple drug molecules to a targeting agent is difficult to accomplish however, without impairing the target specificity of the targeting agent or the activity of the drug. Drug carrier intermediates are frequently employed, whereby a plurality of drug molecules is bound to the intermediate, and the intermediate is bound to the targeting moiety. In this fashion, multiple drug molecules may be associated with a single targeting agent without impairing the specificity of the targeting agent. Human serum albumin (HSA) and other molecules have been used as intermediate drug carriers. U.S. Pat. Nos. 4,507,234; 4,046,722; 4,699,784; and Garrett et al., Int. J. Lancer 31:661-670 (1983), describe immunoconjugates in which a drug carrier is interposed between the active moieties and targeting agents to provide higher drug loading.
HSA has also been suggested as a targeting agent for bone imaging agents comprising diagnostic radionuclides to facilitate delivery of the diagnostic agent to soft tissues and blood pool visualization. Diagnostic imaging agents optionally including HSA are taught in U.S. Pat. Nos. 4,440,738, 4,510,125 and 4,707,353.
HSA has been employed as a stabilizing agent for various compositions intended for in vivo administration. For example, U.S. Pat. No. 4,812,557 teaches use of HSA for stabilizing interleukin-2 (IL-2) compositions during storage, freezing, and/or lyophilization. HSA has also been utilized as a stabilizer for radio-iodinated compositions to prevent the loss of immunoreactivity and to protect and stabilize therapeutic quantities of radio-iodinated monoclonal antibody preparations. In these latter applications, HSA is present in solution, but it is not linked to an active or targeting moiety.